This program aims to develop a novel method for cryopreserving human testicular tissue and whole testes, which can also be applied to ovarian tissue and gonadal preservation and ultimately any cells, tissues, and whole organs. This multi-pronged approach builds on recent advances in machine perfusion, nature-inspired cytoprotection strategies, and non-toxic cryoprotectant solutions, combining them with our novel method for constant-volume, pressure-assisted cooling. Our method promises to achieve vitrification of living tissues without the aid of toxic concentrations of cryoprotectants, in a system at thermodynamic equilibrium and potentially at temperatures as high as -80C (capable of dry-ice shipment). It ameliorates or entirely circumvents many of the limitations of conventional cryopreservation methods for large tissues, including damaging ice crystallization, deleterious changes in solute concentrations, and volumetric changes. This approach has the potential to enable indefinite banking of testicular tissues, whole testes, and other living materials while dramatically limiting tissue injury currently associated with ex-vivo storage. The technical objective of this Phase 1 proposal is to develop an optimized cocktail and protocol for preservation and indefinite banking of testicular tissue. We will develop our method across three specific aims. Building upon preliminary studies that demonstrate the feasibility of isochoric preservation, in Aim 1 we will begin with extended feasibility studies focused on investigating the combined effects of temperature, pressure and CPA on testicular-cell viability following equilibrium ice-free isochoric preservation. Specifically, we will use semi- high-throughput screening to measure the viability of human Sertoli cells, Leydig cells and endothelial cells with targeted pressures of up to 160 MPa (pressure at which red-blood cells show improved cryopreservation). In parallel studies in Aim 2, we will use well established methods developed by our broader group to define new thermodynamic profiles for novel, nature-inspired cryoprotectant solutions in an isochoric (constant- volume) system; this will allow us to select solutions that allow for pressure-assisted vitrification of living materials at low to intermediate pressures and much higher storage temperatures than traditionally needed for ice-free cryopreservation. In Aim 3, we select the most effective cryoprotectant solutions established in Aim 1-2 to evaluate our ?high sub-zero vitrification? in human testicular tissue strips and compare outcome with conventional state-of-the-art isobaric preservation. Tissue will be evaluated by measuring tissue-viability, as well as histology, immunohistochemistry and xenotransplantation into infertile nude mice. We will additionally test the augmentation of these methods with subnormothermic machine perfusion, enabling further reduction of ischemic tissue injury. Success of these novel approaches individually or in combination will likely enable breakthroughs in oncofertility and biopreservation research and clinical practice.